Longitudinal analysis reveals age-related changes in the T cell receptor repertoire of human T cell subsets.

A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRß repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier-based (UMI-based) RNA-seq method. Thorough analysis of 1.9 × 108 T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 × 108. Alterations of the TCR repertoire with age were observed in all 4 subsets of T cells. The greatest reduction was observed in naive CD8+ T cells, while the greatest clonal expansion was in memory CD8+ T cells, and the highest increased retention of TCR sequences was in memory CD8+ T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for CD8+ than CD4+ T cells, suggesting that aging has a more profound effect on cytotoxic as opposed to helper T cell functions. This may explain the increased susceptibility of older adults to novel infections.

TCRß repertoire of CD4+ and CD8+ T cells is distinct in richness, distribution, and CDR3 amino acid composition.

The TCR repertoire serves as a reservoir of TCRs for recognizing all potential pathogens. Two major types of T cells, CD4(+) and CD8(+), that use the same genetic elements and process to generate a functional TCR differ in their recognition of peptide bound to MHC class II and I, respectively. However, it is currently unclear to what extent the TCR repertoire of CD4(+) and CD8(+) T cells is different. Here, we report a comparative analysis of the TCRß repertoires of CD4(+) and CD8(+) T cells by use of a 5' rapid amplification of cDNA ends-PCR-sequencing method. We found that TCRß richness of CD4(+) T cells ranges from 1.2 to 9.8 × 10(4) and is approximately 5 times greater, on average, than that of CD8(+) T cells in each study subject. Furthermore, there was little overlap in TCRß sequences between CD4(+) (0.3%) and CD8(+) (1.3%) T cells. Further analysis showed that CD4(+) and CD8(+) T cells exhibited distinct preferences for certain amino acids in the CDR3, and this was confirmed further by a support vector machine classifier, suggesting that there are distinct and discernible differences between TCRß CDR3 in CD4(+) and CD8(+) T cells. Finally, we identified 5-12% of the unique TCRßs that share an identical CDR3 with different variable genes. Together, our findings reveal the distinct features of the TCRß repertoire between CD4(+) and CD8(+) T cells and could potentially be used to evaluate the competency of T cell immunity.

Development of an immunochromatographic test strip for detection of cholera toxin.

Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.

Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection.

An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.

Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-beta.

Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A(2), are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A(2) synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-kappabeta signaling pathway.

Proteasome inhibitors enhance endothelial thrombomodulin expression via induction of Krüppel-like transcription factors.

OBJECTIVE: Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathological thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We investigated the effects of proteasome inhibitors on endothelial thrombomodulin expression and function. METHODS AND RESULTS: Proteasome inhibitors as a class markedly induced the expression of thrombomodulin and enhanced the protein C activating capacity of endothelial cells. Thrombomodulin upregulation was independent of NF-kappaB signaling, a principal target of proteasome inhibitors, but was instead a direct consequence of increased expression of the Krüppel-like transcription factors, KLF2 and KLF4. These effects were confirmed in vivo, where systemic administration of a proteasome inhibitor enhanced thrombomodulin expression that was paralleled by changes in the expression of KLF2 and KLF4. CONCLUSIONS: These findings identify a novel mechanism of action of proteasome inhibitors that may help to explain their clinically observed thromboprotective effects.

Regulation of epithelial junctions by proteins of the ADP-ribosylation factor family.

ADP- ribosylation factor (ARF) proteins play a pivotal role in the regulation of membrane traffic and the organization of the cytoskeleton that are crucial to fundamental cellular processes, such as intracellular sorting/trafficking of newly synthesized proteins and endocytosis/exocytosis. In epithelial junctions, the ARF proteins are intimately associated with the dynamics of transmembrane proteins, such as E- cadherin and beta- 1 integrin, and the adaptor proteins such as paxillin. In addition, ARF proteins play a key regulatory role in the remodeling of actin cytoskeleton necessary for the formation of membrane ruffles and protrusions in association with phospholipase D and members of the Rho GTPase family. These activities of ARF proteins influence not only the formation, stability and functional integrity of epithelial junctions but also the cell motility as well. In this review, I have attempted to provide a compendium of evidence that has contributed to our evolving understanding of these ARF proteins as well as their regulators (guanine nucleotide exchange factors and GTPase- activating proteins) in the regulation of epithelial junctions. In addition, I have also highlighted potential mechanisms as to how these intricate regulatory pathways are regulated both spatially and temporally.

cAMP-dependent protein kinase A (PKA) signaling induces TNFR1 exosome-like vesicle release via anchoring of PKA regulatory subunit RIIbeta to BIG2.

The 55-kDa TNFR1 (type I tumor necrosis factor receptor) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-bromo-cyclic AMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a protein kinase A (PKA)-dependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIbeta modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIbeta to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIbeta to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide exchange protein that activates class I ADP-ribosylation factors and as an AKAP for RIIbeta that localizes PKA signaling within cellular TNFR1 trafficking pathways.

Effect of psychotropic drugs on the 21-hydroxylation of neurosteroids, progesterone and allopregnanolone, catalyzed by rat CYP2D4 and human CYP2D6 in the brain.

We determined the effects of psychotropic drugs on the cytochrome P450 2D (CYP2D)-mediated 21-hydroxylation of progesterone (PROG) and allopregnanolone (ALLO) with the goal of clarifying whether neurosteroid levels are affected by psychotropic drugs in the brain. PROG or ALLO was incubated with rat CYP2D4 or human CYP2D6 in the presence of typical psychotropic drugs, fluoxetine, imipramine, desipramine, mazindol, and GBR12909, and the 21-hydroxylated metabolites of PROG and ALLO were determined by high performance liquid chromatography and liquid chromatography-tandem mass spectrometry, respectively. Fluoxetine competitively inhibited CYP2D4-mediated PROG 21-hydroxylation and increased both Km and Vmax values of CYP2D6-mediated PROG 21-hydroxylation. In addition, fluoxetine competitively inhibited ALLO 21-hydroxylation mediated by CYP2D4 and CYP2D6. Imipramine, desipramine, mazindol, and GBR12909 competitively inhibited PROG 21-hydroxylation mediated by CYP2D4 and/or CYP2D6, and all psychotropic drugs inhibited ALLO 21-hydroxylation mediated by CYP2D4 and/or CYP2D6. The inhibition constants (Ki values) of imipramine, desipramine, and mazindol against the 21-hydroxylation of PROG and ALLO by CYP2D6 were lower than those by CYP2D4. These results indicate that psychotropic drugs including fluoxetine affected the metabolism of neurosteroids, such as PROG and ALLO in the brain, suggesting that the regulation of the neurosteroid levels is modified by central nervous system-active drugs that inhibit brain CYP2D isoforms.

Over-expression of protein disulfide isomerase reduces the release of growth hormone induced by bisphenol A and/or T3.

We previously isolated a bisphenol A (BPA)-binding protein from rat brain and showed that it was identical to the protein disulfide isomerase (PDI), which also serves as a 3,3',5-triiodo-l-thyronine (T3)-binding protein. In this study, we investigated the effects of BPA on the production of growth hormone (GH) in GH3 cells and examined the possible involvement of PDI in this process. When administered singly, BPA and T3 each induced GH release in GH3 cells. When cells were treated with the combination of BPA and T3, the release of GH was much greater than that by T3 alone, but GH mRNA and promoter activity were not increased. These results suggested that the synergistic effect of T3 plus BPA on GH release was due to posttranslational regulation. Over-expression of PDI suppressed GH mRNA expression and GH release, suggesting that PDI modulates the T3-induced gene expression. We conclude that BPA can disrupt the thyroid hormone function in GH3 cells, and GH release induced by T3 is influenced by expression of PDI.

The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles.

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

GEP100/BRAG2: activator of ADP-ribosylation factor 6 for regulation of cell adhesion and actin cytoskeleton via E-cadherin and alpha-catenin.

GEP(100) (p100) was identified as an ADP-ribosylation factor (ARF) guanine nucleotide-exchange protein (GEP) that partially colocalized with ARF6 in the cell periphery. p100 preferentially accelerated guanosine 5[gamma-thio]triphosphate (GTPgammaS) binding by ARF6, which participates in protein trafficking near the plasma membrane, including receptor recycling, cell adhesion, and cell migration. Here we report that yeast two-hybrid screening of a human fetal brain cDNA library using p100 as bait revealed specific interaction with alpha-catenin, which is known as a regulator of adherens junctions and actin cytoskeleton remodeling. Interaction of p100 with alpha-catenin was confirmed by coimmunoprecipitation of the endogenous proteins from human HepG2 or CaSki cells, although colocalization was difficult to demonstrate microscopically. alpha-Catenin enhanced GTPgammaS binding by ARF6 in vitro in the presence of p100. Depletion of p100 by small interfering RNA (siRNA) treatment in HepG2 cells resulted in E-cadherin content 3-fold that in control cells and blocked hepatocyte growth factor-induced redistribution of E-cadherin, consistent with a known role of ARF6 in this process. F-actin was markedly decreased in normal rat kidney (NRK) cells overexpressing wild-type p100, but not its GEP-inactive mutants, also consistent with the conclusion that p100 has an important role in the activation of ARF6 for its functions in both E-cadherin recycling and actin remodeling.

Bisphenol A binds to protein disulfide isomerase and inhibits its enzymatic and hormone-binding activities.

Bisphenol A [2,2-bis-(4-hydroxyphenyl) propane; BPA] is a versatile industrial material for plastic products, but is increasingly being recognized as a pervasive industrial pollutant as well. Accumulating evidence indicates that the environmental contaminant BPA is one of the endocrine-disrupting chemicals that potentially can adversely affect humans as well as wildlife. To define the molecular aspects of BPA action, we first investigated the molecules with which it physically interacts. High BPA-binding activity was detected in the P2 membrane fraction prepared from rat brains. As determined by SDS-PAGE analysis, the molecular mass of a BPA-binding protein purified from the rat brain P2 fraction was 53 kDa. The N-terminal amino acid sequence of the purified BPA-binding protein was identical with that of the rat protein disulfide isomerase (PDI), which is a multifunctional protein that is critically involved in the folding, assembly, and shedding of many cellular proteins via its isomerase activity in addition to being considered to function as an intracellular hormone reservoir. The Kd value of BPA binding to recombinant rat PDI was 22.6 +/- 6.6 microm. Importantly, the binding activity of L-T3 and 17beta-estradiol hormones to PDI was competitively inhibited by BPA in addition to abolishing its isomerase activities. In this paper we report that the ubiquitous and multifunctional protein PDI is a target of BPA and propose that binding to PDI and subsequent inhibition of PDI activity might be mechanistically responsible for various actions of BPA.

A new epitope of CYP2D6 recognized by liver kidney microsomal autoantibody from japanese patients with autoimmune hepatitis.

Liver-kidney microsomal antibodies type 1 (LKM-1) are a diagnostic marker for autoimmune hepatitis type II (AIH-II). However, LKM autoantibodies are also detected in a small percentage of patients with chronic hepatitis C. The autoantigen to anti-LKM-1 has been identified to be CYP2D6. To identify the specific antigenic site of CYP2D6 for LKM-1 serum, we established an ELISA with peptides spanning the entire sequence of CYP2D6. Human CYP2D6 containing histidine tag was expressed in Escherichia coli. Purified CYP2D6 was digested by lysyl endopeptidase. The linker including the histidine tag has a lysine residue in its C-terminal and can be removed by digestion. Digested peptides were separated by reversed-phase HPLC and coated on ELISA plates chemically with glutaraldehyde. The immunoreactivity of two LKM-1-positive sera (HCV-negative) and five HCV-positive sera from Japanese patients was investigated with the plates. These sera recognized peptides 1-146, 181-214, 246-281, 284-391, and 412-429. The peptide 1-146 was recognized by LKM-1-positive sera but not HCV-positive sera and is a new epitope found in this study. Seven short peptides spanning peptide 1-146 were synthesized and ELISAs were conducted with these peptides. However, two sera recognized none of these peptides, suggesting that two LKM-1-positive sera recognize the conformational immunogenic site of peptide 1-146.

Inhibitory effects of environmental chemicals on protein disulfide isomerase in vitro.

BACKGROUND: The influence of endocrine disrupting chemicals (EDCs) upon physiological functions and their mechanisms is of concern. We have previously demonstrated that protein disulfide isomerase (PDI) was a target molecule of bisphenol A (BPA), which is considered to be EDC. PDI plays a key role in protein folding as an isomerase and also possesses a 3,3',5-triiodo-L-thyronine (T3)-binding activity. Since PDI activities were inhibited by BPA in our previous study, BPA might have adverse effects on physiological functions via the inhibition of PDI activities. We conducted this study to identify the compounds which disturb PDI activities as well as BPA, and to discuss their structural characteristics. METHODS: We examined the effects of 22 suspected EDC on both the T3-binding activity and isomerase activity of rat recombinant PDI. RESULTS: Among the 22 compounds, only phenolic compounds, namely BPA, p-octylphenol, p-nonylphenol, 2,4-dichlorophenol, pentachlorophenol, tetrabromobisphenol A, and tetrachlorobisphenol A, inhibited T3 binding to PDI. Furthermore non-halogenated compounds among these phenolic compounds, such as BPA, p-octylphenol, and p-nonylphenol, showed inhibitory effects on the isomerase activity of PDI. CONCLUSIONS: Our results suggest that phenolic groups might have important inhibitory effects on the T3-binding activity of PDI, and that compounds with phenolic groups might have the same effects on PDI. Furthermore, non-halogenated phenolic compounds had inhibitory effects on the isomerase activities of PDI in addition to T3-binding activity, indicating that these compounds might also have adverse effects on protein folding, which PDI participates in by catalyzing rearrangements of disulfide bonds as an isomerase.

Effect of genetic polymorphism on the metabolism of endogenous neuroactive substances, progesterone and p-tyramine, catalyzed by CYP2D6.

Metabolic activities toward endogenous substrates in the brain, progesterone and p-tyramine, by cytochrome P450 2D6.2 (CYP2D6.2), CYP2D6.10A, CYP2D6.10C, and P34S, G42R, R296C, and S486T mutants expressed in recombinant Saccharomyces cerevisiae were compared with those by CYP2D6.1 (wild-type) in order to clarify the effects of genetic polymorphism of CYP2D6 on the metabolism of neuroactive steroids and amines in the brain. For the 6beta-hydroxylation of progesterone, the V(max) values for CYP2D6.2, CYP2D6.10A, and the P34S and G42R mutants, were less than half of those for CYP2D6.1, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type. The V(max)/K(m) values for CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were 12-31% of that for CYP2D6. The 16alpha-hydroxylation and 21-hydroxylation of progesterone by CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were not detected, and the R296C mutant had a higher K(m) for the 16alpha-hydroxylation and a lower V(max) for the 21-hydroxylation than those for CYP2D6.1. For dopamine formation from p-tyramine, the K(m) values for CYP2D6.2 and the R296C mutant were higher than those for CYP2D6.1, CYP2D6.10A, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type. The V(max)/K(m) values for CYP2D6.2, CYP2D6.10A, CYP2D6.10C and the P34S, G42R and R296C mutants were less than 45% of those for the wild-type. These results suggest the possibility that the polymorphism of CYP2D6, including CYP2D6*2, CYP2D6*10 and CYP2D6*12, might affect an individual behavior and the central nervous system through endogenous compounds, such as neuroactive steroids and tyramine, in the brain.

Role of phenobarbital-inducible cytochrome P450s as a source of active oxygen species in DNA-oxidation.

We investigated the biological effects of the active oxygen produced by P450s. First, we identified which isoforms of P450 efficiently produced active oxygen using electron spin resonance. Eight forms of P450 purified from rat liver were used. Of these, CYP1A2, 2B1, 2C11 and 3A2 produced hydroxyl radicals efficiently. Phenobarbital (PB) which is a typical inducer of CYP2B1 and 3A2 induced production of hydroxyl radicals by rat liver and ketoconazole, an inhibitor of P450, inhibited production of hydroxyl radicals in vitro. PB is a tumor promoter as well as the P450-inducer. We investigated oxidation of the genomic DNA by the hydroxyl radicals produced by PB-inducible P450 in vitro and in vivo. 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of DNA oxidation in vivo was assayed by HPLC. PB strongly induced the production of 8-OHdG in the rat liver. While ketoconazole inhibited the production of 8-OHdG in vivo. These results suggest that active oxygen produced by P450 oxidized genomic DNA and induction of P450 increased oxidative stress that may contribute to tumor initiation and promotion.

Cytochrome P450 2D catalyze steroid 21-hydroxylation in the brain.

mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17alpha-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative gamma-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC(50) value of 2 microm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.

A novel transcriptional element which regulates expression of the CYP2D4 gene by Oct-1 and YY-1 binding.

We first identified the transcriptional regulatory element of the CYP2D4 gene. CYP2D4 is of interest in brain pharmacology and physiology because this enzyme can be involved in the metabolism of endogenous and exogenous compounds, which act on the central nervous system. Transfection studies using a series of the CYP2D4 promoter luciferase constructs identified the transcriptional element of CYP2D4 in the sequence between nucleotides -116 and -90 (named the neural expression regulatory element, NERE). The nucleotide sequence of NERE was specific for the CYP2D4 gene. Within this region, two nuclear factor-binding sequences, Oct-1 and YY-1, were present. Oct-1 acts as the activator of the CYP2D4. The core sequence of the YY-1 binding motif partially overlapped that of the Oct-1 binding motif. YY-1 may act as the repressor of CYP2D4, which interferes with Oct-1 activation by its binding to NERE. We concluded that a novel transcriptional regulatory element NERE specifically regulates the expression of the CYP2D4. This regulation system may be involved in the unique distribution of this isoform, such as the expression in the brain.

Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities.

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC(50) values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K(m) and V(max) values ranging between 78 and 145 microM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC(50) values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC(50) values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC(50) values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.